Quartz Crystal Microbalance as a Holistic Detector for Quantifying Complex Organic Matrices during Liquid Chromatography: 1. Coupling, Characterization, and Validation

A matrix in highly complex samples can cause adverse effects on the trace analysis of targeted organic compounds. A suitable separation of the target analyte(s) and matrix before the instrumental analysis is often a vital step for which chromatographic cleanup methods remain one of the most frequently used strategies, particularly high-performance liquid chromatography (HPLC). The lack of a simple real-time detection technique that can quantify the entirety of the matrix during this step, especially with gradient solvents, renders optimization of the cleanup challenging. This paper, along with a companion one, explores the possibilities and limitations of quartz crystal microbalance (QCM) dry-mass sensing for quantifying complex organic matrices during gradient HPLC. To this end, this work coupled a QCM and a microfluidic spray dryer with a commercial HPLC system using a flow splitter and developed a calibration and data processing strategy. The system was characterized in terms of detection and quantification limits, with LOD = 4.3–15 mg/L and LOQ = 16–52 mg/L, respectively, for different eluent compositions. Validation of natural organic matter in an environmental sample against offline total organic carbon analysis confirmed the approach’s feasibility, with an absolute recovery of 103 ± 10%. Our findings suggest that QCM dry-mass sensing could serve as a valuable tool for analysts routinely employing HPLC cleanup methods, offering potential benefits across various analytical fields.

S1 Chemicals, Materials, Standard Solutions, and Spray Fabrication Table S1 List of reagents, solvents, and analytical standards.
The microfluidic spray-dryers were fabricated in-house at the Heinz Nixdorf-Chair of Biomedical Electronics at the Center for Translational Cancer Research of the Technical University of Munich (TranslaTUM).Fast and reliable production of microfluidic spray-dryers was achieved using a two-layer soft lithography approach according to a previously published protocol 1 .Microfluidic channels were designed in AutoCAD 2021 (Autodesk GmbH).Negative resists SU8-3025 and SU8-3050 (MicroChem Corp.) were spin-coated and patterned using a maskless laser lithography system (Dilase 250, Kloe, France) on a 3" Si substrate to obtain the negative master mold.The first layer has a thickness of 20 µm and contains the channel for the liquid sample, while the second layer has a thickness of 70 µm and contains the channel for the gas.PDMS (Sylgard 184, Dow Corning) was mixed at a ratio of 9:1 (w/w), degassed, and cured in the negative SU8 master mold for 60 min at 65 °C.Microfluidic devices were cut along alignment marks using a razor blade, and holes for tubings were punched using a 0.5 mm biopsy punch (World Precision Instruments).Individual PDMS devices were cleaned using isopropanol and acetone and blown dry with nitrogen.PDMS devices were activated using oxygen plasma for 60 s at 30 W (Zepto, Diener electronic GmbH, Germany).A drop of deionized water was applied to the surface to facilitate the alignment of two PDMS devices under a stereo microscope for the final microfluidic device.A permanent bond between PDMS parts is formed by curing the assembled devices for 60 min at 85 °C.The cross-section of the channels for liquid and gas delivery were measured to be 27 × 20 µm 2 (w × h) and 110 × 70 µm 2 (w × h), respectively.

S2 Determination of Split Ratios
Split ratios (R split ) were determined for different Vernier scale settings (56, 66, 73, 79, 94, and 112) for three different CH 3 OH/H 2 O mobile phase compositions [85/15, 50/50, and 15/85 (v/v)] by spraying the mobile phase containing 500 mg/L NaCl for 30 min into a vial.The dried salt was reconstituted in 8 mL H 2 O.The salt concentration in solution was determined by measuring the salinity using a salinometer (MultiLine F/SET-3, WTW, Germany).
Dividing the corrected salinity (κ corrected ) by the molar conductivity of NaCl (Λ N aCl m ) results in the concentration of the sample in the vial (c sample ) (see Eq S1 and S2).
The flow to the spray-dryer (Q spray ) was calculated using Eq S3 by dividing the sprayed mass (m sample ), calculated using c sample , by the sprayed time (t) and by the concentration of salt (C N aCl ) in the mobile phase.
The split ratio (R split ) was calculated using Eq S4.Here, the high flow, which is the difference of the input flow (Q input ) and the low flow (Q spray ), is divided by the low flow.

S3 Microfluidic Spray Efficiency of Drying Different Solvents
To ensure that the sprayed sample on the QCM fully dries, especially with gradient elution, a mass was deposited for 10-15 min from three different solvent compositions

S5 TOC Validation
Flow chart of offline TOC measurement of HPLC fractions.The mobile phase is split using a post-column adjustable flow splitter.The high flow goes to a fraction collector; each fraction is collected for 30 seconds (volume: 0.25 mL).The fraction is evaporated and reconstituted in 16 mL H 2 O and measured using TOC analysis.

Figure S1 Figure S2 Figure
Figure S1Dry mass sensing on QCM using microfluidic spray in different eluent compositions associated with frequency shift starting from 0 min.The orange dashed vertical line denotes a complete shut down of eluent flow whilst maintaining the flow of drying nitrogen gas.

3 Figure S5
Figure S5 Frequency raw data (blank = black, sample = blue, calibration = red) of the QCM measurement during the TOC validation experiment.The grey bar shows the measurement window (2.5 min dead time).

GRADIENT
figure hold on for i = blank_index

Table S2
HPLC gradient conditions for the TOC validation measurement.